Experimentation using human cells is generally desired to study human diseases. In particular, studies of diseases, in which many drug-metabolizing enzymes confirmed to have species specificity, viruses, the hosts of which are limited to humans, and the like are involved, require to use human cells, and particularly human hepatocytes. However, the supply of human hepatocytes is limited and in vitro proliferation of human hepatocytes while keeping their differentiation status is very difficult. The use of in vivo environment is relatively efficient for the proliferation of human hepatocytes. Specifically, a gene accelerating the death of mouse hepatocytes is introduced into mice that have been produced from immunodeficient mice as the genetic background to produce transgenic mice, human hepatocytes are transplanted into the transgenic mice, and then human hepatocytes are proliferated. In this manner, the replacement of most mouse hepatocytes by human hepatocytes has been attempted.
Liver disease caused by the infection of human liver with viruses is a disease difficult to treat in recent years in medical practice. Animal species susceptible to these viruses that infect human hepatocytes are limited to humans and chimpanzees. Tests using human hepatocytes are required to develop remedies against these viral infections. Also, hepatocytes play important roles in drug metabolism. Elucidation of the metabolic pathways of individual drugs in humans is considered to lead to the development of new pharmaceutical products. However, species specificity is present in many drug-metabolizing enzymes, and thus elucidation of the drug metabolic pathways in humans requires to conduct tests using human hepatocytes.
Regarding Hepatitis C virus (HCV), about 1500,000 carriers of Hepatitis C virus (HCV carriers), and about 400,000 to 500,000 patients other than these carriers are estimated to be treated in Japan. The number of chronic hepatitis C patients receiving interferon administration is said to be annually 30,000 to 40,000. In these days, new antiviral agents targeting various sites of viral genome are under development. However, the advancement thereof is significantly inhibited because of the lack of reliable HCV animal models with high reproducibility. This can be said for not only HCV, but also other types of viral hepatitis such as hepatitis type B virus (HBV). Hosts for these viruses are only humans and chimpanzees. Therefore, development of small model animals produced by replacing human hepatocytes by a host's hepatocytes is desired for large-scale development and study of antiviral agents using animals.
Fatty liver is developed due to the accumulation of neutral fat in the liver. In recent years, the incidence of non-alcoholic steatohepatitis (NASH) that is hepatitis resulting from the accumulation of fat in the liver is increasing. This disease may proceed to diseases with poor prognosis such as chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Meanwhile, the absence of effective remedies against such liver diseases has been suggested (Non-patent Literature 1). The development of such remedies also requires the presence of optimum animal models.
If the use of model animals having human hepatocytes as a result of replacement becomes possible for the study of the above diseases, this will contribute to many studies for drug development. However, the preparation of the model animals requires efficient proliferation of human hepatocytes after transplantation thereof into host animals and successful replacement thereof by the host's hepatocytes.
Several examples of transplantation of human hepatocytes into transgenic mice have been reported, wherein human hepatocytes are transplanted into the transgenic mice in which an urokinase-type plasminogen activator (hereinafter, referred to as “uPA”) gene is expressed liver-specifically, so as to damage mouse hepatocytes. uPA transgenic mice prepared using the genomic sequence of uPA (Non-patent Literature 2) and uPA transgenic mice prepared using the cDNA of uPA (Non-patent Literature 3) have been reported. All of these uPA transgenic mice are required to have the uPA gene in a homozygous form, since the engraftment of transplanted human hepatocytes is difficult when the mice have the uPA gene in a heterozygous form. However, the preparation of transgenic mice having the uPA gene in a homozygous form requires at least two generations and at least 6 months. Moreover, homozygous mice are obtained in a proportion of about only 25% with respect to the total number of the thus obtained mice. It has been difficult to prepare transgenic mice having a large quantity of the uPA gene in a homozygous form within a short period. It has also been difficult to prepare a cross-bred line with another transgenic mouse due to a similar reason. Moreover, in transgenic mice produced using a conventional uPA genomic sequence, the recombination of the uPA gene introduced into the liver takes place over time, and the loss of the uPA gene is observed. Since mouse cells lacking the uPA gene regenerate hepatocytes again, it has been difficult for human hepatocytes to engraft after transplantation thereof into heterozygous mice. Furthermore, in homozygous mice, mouse hepatocytes are regenerated due to the loss of the uPA gene, and thus a gradual decrease in human hepatocytes that have engrafted is frequently observed among mice. Hence, uPA transgenic mice that can be produced efficiently in large quantity and enables easy preparation of a cross-bred line with another transgenic mouse have been desperately desired in the art.